The diverse repertoire of antigen receptors required for mammalian immunity is generated in precursor lymphocytes by V(D)J recombination, which fuses random combinations of immunoglobulin (Ig) or T cell receptor (Tcr) gene segments to form a signature variable region exon for each lymphocyte. Although V(D)J recombination is critical for diversification of antigen receptors, reshaping the genome of somatic cells is perilous. Errant targeting of V(D)J recombination produces chromosomal translocations involving proto- oncogenes, which initiate most known leukemias and lymphomas. Moreover, V(D)J recombination must be sequentially activated or repressed at distinct regions within Ig and Tcr loci to orchestrate lymphocyte development. In large part, the tissue-, stage- and allele-specific control of V(D)J recombination is coordinated by genetic elements that alter the accessibility of chromatin at Ig and Tcr loci. The applicant's laboratory has shown that transcriptional promoters and enhancers serve as accessibility control elements (ACEs) to direct the first step of Tcrb gene assembly (D2AEJ2 recombination). Studies in the prior funding period revealed that ACEs mediate a series of biochemical events that culminate in a recombinase-accessible Tcrb locus, including (i) enhancer-directed chromatin opening, (ii) formation of a higher order promoter-enhancer complex, and (iii) promoter-dependent recruitment of chromatin remodeling complexes to unmask neighboring gene segments for recombination. Based on these discoveries, we hypothesize that thymocyte development relies on a sequential recruitment of transcription factors to ACEs within Tcrb, which coordinate a precise epigenetic program leading to regional or highly localized changes in recombinase accessibility. To test this central hypothesis, we will identify TFs that bridge distal ACEs to generate a promoter-enhancer holocomplex (Aim 1). Aim 2 studies will establish the molecular mechanisms by which chromatin remodeling complexes are recruited to ACEs as well as their subsequent mode of action to confer recombinase accessibility. Aim 3 studies will follow, in real-time, the contingent mechanisms that control stepwise opening of Tcrb to recombinase. In Aim 4, we will define epigenetic changes in nucleosome composition and histone modifications that protect specific gene segments from the long-range ACE function of enhancers and subsequently unlock these segments for recombination. Overall, the project will establish the genetic, epigenetic, and topological mechanisms by which V(D)J recombinase is permitted access to specific gene segments, resulting in a fully functional immune repertoire. The research will also provide insights into processes that control chromatin accessibility during transcription, replication, and recombination of select genomic information in all eukaryotic cells. Public Health Relevance: Developing lymphocytes cut-and-paste together an enormous collection of antigen receptor genes by the process of V(D)J recombination. This unique process of DNA recombination must be tightly regulated in precursor cells to guide their development into functional, mature lymphocytes and to preclude chromosomal translocations that lead to lymphoid tumors. We propose studies to address how precursor T lymphocytes activate the T cell receptor beta locus at the earliest stages of thymocyte development to ensure its proper assembly by V(D)J recombination.